Organic compounds

ABSTRACT

The invention relates to polypeptides and modified polypeptides derived from fibrinogen comprising one of the following sequences: 
         X 1 FLAEX 6 X 7 X 8 V    DX 2 LAEX 6 X 7 X 8 V    DFX 3 AEX 6 X 7 X 8 V    DFLX 4 EX 6 X 7 X 8 V    DFLAX 5 X 6 X 7 X 8 V    DFLAEX 6 X 7 X 8 V    DFLAEX 6 X 7 X 8 X 9  wherein X is any amino acid residue and which polypeptide has anti-angiogenic activity.

The invention relates to the anti-angiogenic polypeptides and modifiedpolypeptides derived from fibrinogen including oligomers of saidpolypeptides.

Angiogenesis, the development of new blood vessels from an existingvascular bed, is a complex multistep process that involves thedegradation of components of the extracellular matrix and then themigration, proliferation and differentiation of endothelial cells toform tubules and eventually new vessels. Angiogenesis is important innormal physiological processes including, by example and not by way oflimitation, embryo implantation; embryogenesis and development; andwound healing. Excessive angiogenesis is also involved in pathologicalconditions such as tumour cell growth and non-cancerous conditions suchas neovascular glaucoma, rheumatoid arthritis, psoriasis and diabeticretinopathy.

The vascular endothelium is normally quiescent. However, upon activationendothelial cells proliferate and migrate to form microtubules whichwill ultimately form a capillary bed to supply blood to developingtissues and, of course, a growing tumour. A number of growth factorshave been identified which promote/activate endothelial cells to undergoangiogenesis. These include, by example and not by way of limitation;vascular endothelial growth factor (VEGF); transforming growth factor(TGFb); acidic and basic fibroblast growth factor (aFGF and bFGF); andplatelet derived growth factor (PDGF) (1,2).

VEGF is a an endothelial cell-specific growth factor which has a veryspecific site of action, namely the promotion of endothelial cellproliferation, migration and differentiation. VEGF is a dimeric complexcomprising two identical 23 kD polypeptides. The monomeric form of VEGFcan exist as four distinct polypeptides of different molecular weight,each being derived from an alternatively spliced mRNA. Of the fourmonomeric forms, two exist as membrane bound VEGF and two are soluble.VEGF is expressed by a wide variety of cell/tissue types includingembryonal tissues; proliferating keratinocytes; macrophages; tumourcells. Studies (2) have shown VEGF is highly expressed in many tumourcell-lines including glioma and AIDS-associated Kaposi's sarcoma. VEGFactivity is mediated through VEGF specific receptors expressed byendothelial cells and tumour cells. Indeed, VEGF receptors areup-regulated in endothelial cells which infiltrate tumours therebypromoting tumour cell growth.

bFGF is a growth factor which functions to stimulate the proliferationof fibroblasts and endothelial cells. bFGF is a single polypeptide chainwith a molecular weight of 16.5 Kd. Several molecular forms of bFGF havebeen discovered which differ in the length at their amino terminalregion. However the biological function of the various molecular formsappears to be the same. bFGF is produced by the pituitary gland and isencoded by a single gene located on human chromosome 4.

A number of endogenous inhibitors of angiogenesis have been discovered,examples of which are angiostatin and endostatin, which are formed bythe proteolytic cleavage of plasminogen and collagen XVIII respectively.Both of these factors have been shown to suppress the activity ofpro-angiogenic growth factors such as vascular VEGF and bFGF. Both alsosuppress endothelial cell responses to VEGF and bFGF in vitro, andreduce the vascularisation and growth of experimental tumours in animalmodels.

Fibrinogen, the soluble circulating precursor of fibrin, is a dimericmolecule containing pairs of non-identical chains, (ie the α-, β- andγ-chains). These are arranged as three discrete domains, the two outerD-domains and the central E domain (4). Fibrinogen can be digestedeither by plasmin or thrombin.

The first step in plasmin cleavage of fibrinogen is the cleavage of thea chain C-terminal domain. Plasmin then cleaves the two D domains fromthe one E domain (consisting of the NH2 terminal regions of the α-, β-and γ-chains held together by disulphide bonds) and numerous smallerfragments including a small peptide, beta1-42 (amino terminal of theβ-chain) (5). Thrombin, on the other hand, produces a fibrin monomer andtwo copies of fibrinopeptides A and B (4). Fibrinogen has been shown toaccumulate around leaky blood vessels in solid tumours (5), Fibrinogenhas also been shown to polymerise at host-tumour interface to formfibrin networks that promote tumour angiogenesis by supporting theadhesion, migration, proliferation and differentiation of endothelialcells (7).

The fibrin E-fragment (FnE-fragmert), produced by the proteolyticcleavage of fibrin, stimulates angiogenesis in the chorioallantoicmembrane assay (8). Furthermore, the amount of this protein present ininvasive breast carcinomas positively correlates with the degree oftumour vascularity (5).

A potent, new inhibitor of angiogenesis, which is a 50 kDa proteolyticfragment of fibrinogen, fibrinogen E, is disclosed in our co-pendingpublished application, WO01/88129. We have also identified a domainwithin the fibrinogen E fragment which has the same anti-angiogenicactivity as the very much larger fibrinogen E fragment. The domain islocated at the amino terminus of the α chain and is referred to as α1-24and is disclosed in our co-pending application WO02/18440. Peptides andpeptide variants derived from the domain have anti-angiogenic activity.The content of WO02/18440, with respect to DNA sequence disclosed inFIG. 5A and the peptides and peptide variants disclosed in FIG. 5B andon page 3, 4 and 5 we hereby disclaim.

Surprisingly, we have now found, in accordance with the presentinvention, that a fragment comprising a short sequence motif of theα1-24 peptide is sufficient for said anti-angiogenic activity.Accordingly, the invention provides in a first aspect a polypeptide of15 or less amino acid residues comprising one of the followingsequences: X₁FLAEX₆X₇X₈V DX₂LAEX₆X₇X₈V DFX₃AEX₆X₇X₈V DFLX₄EX₆X₇X₈VDFLAX₅X₆X₇X₈V DFLAEX₆X₇X₈V DFLAEX₆X₇X₈X₉wherein X is any amino acid residue and which polypeptide hasanti-angiogenic activity. As used herein, X with a subscript numberrefers to a variable amino acid at a specific position, whereas Xwithout a subscript number refers to a variable amino acid residueindependent of its position and may refer to any of the X with subscriptnumber. X may be a proteinogenic or a non-proteinogenic amino acid.

As further described hereinbelow, amino acid residues may be replacedwithout substantially reducing anti-angiogenic activity. Preferably, atleast 90% of the anti-angiogenic activity is retained, more preferablyat least 80%, or at least 70%, or at least 60%, or at least 50% isretained.

In a preferred embodiment of the present invention, X₁ is an acidicamino acid, X₂, X₃, X₄, X₅ are selected from the group consisting ofglycine, alanine, valine, leucine, isoleucine, proline, X₆, X₇, X₈ maybe any amino acid, may be a proteinogenic or a non-proteinogenic aminoacid, and X₉ is a non-polar amino acid. In a particularly preferredembodiment, said polypeptide comprises the sequence DFLAEGGGV.

As will be apparent to one skilled in the art and as further describedhereinbelow, peptides can be modified to improve pharmacologicalproperties by incorporation of “non-proteinogenic” amino-acids withouthaving a deteriorating effect on the biological activity of saidpeptide. Accordingly, in a preferred embodiment of the presentinvention, one or both of amino acid residues X₁ and X₅ aregamma-carboxy-L-glutamic acid, L-2-aminoadipic acid, L-3-aminoadipicacid or (+/−)-lminocyclopentane-cis-1,3-dicarboxylic acid. In additionor alternatively, X₂ is an amino acid residue selected from the groupconsisting of L-2-naphthylalanine,L-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid and4,4′-biphenylalanine. In addition or alternatively, one or more of aminoacid residues X₃, X₄ or X₉ are selected from the group consisting of1-aminocyclopropanecarboxylic acid, 3-aminopentane-3-carboxylic acid,R-2-amino-2-cyclohexyl-propanoic acid.

In another embodiment of the above aspect, the present inventionprovides a polypeptide of 15 or less amino acid residues comprising asequence selected from the group consisting of: X₁X₂X₃X₄X₅X₆X₇X₈X₉RX₁X₂X₃X₄X₅X₆X₇X₈X₉RG X₁X₂X₃X₄X₅X₆X₇X₈X₉RGPwherein X₁, X₂, X₃, X₄, X₅, X₆, X₇, X₈ and X₉, respectively, are definedas above.

In another embodiment of the above aspect, the present inventionprovides a polypeptide of 15 or less amino acid residues comprising asequence selected from the group consisting of: GX₁X₂X₃X₄X₅X₆X₇X₈X₉EGX₁X₂X₃X₄X₅X₆X₇X₈X₉ GEGX₁X₂X₃X₄X₅X₆X₇X₈X₉wherein X₁, X₂, X₃, X₄, X₅, X₆, X₇, X₈ and X₉, respectively, are definedas above.

In accordance with another aspect of the present invention, it was foundthat one or more of amino acid residues X₇, X₈ and X₉ can be replacedwith a flexible chemical linker without without substantially reducinganti-angiogenic activity. Accordingly, the present invention provides apolypeptide, said polypeptide having one or more of amino acid residuesX₇, X₈ and X₉ replaced by a flexible chemical linker. Preferably, thelinker contains a “backbone” with 3 to 40, preferably 4 to 30, morepreferably 5 to 15, most preferably 6 to 12 atoms, linking the flankingamino acids. The backbone atoms are preferably C-atoms, but may alsocontain heteroatoms such as O, N, P or S. The backbone may further besubstituted with alkyl-, aryl- or alkoxy-groups or halogen. Flexiblechemical linkers include by examples and not by way of limitation:—CH₂—CH₂—CH₂)_(x)—, —(CR₁R₂—CR₃R₄)_(x)—, —CH₂CH₂O)_(x)—,—(NH—CR₁R₂—CR₃R₄—CO)_(x)— wherein x=1-10 and wherein R₁, R₂, R₃, R₄, R₅and R₆ are for instance a hydrogen-halogen-, alkyl-, aryl-, arlylalkyl-or alkoxy-radical, may be the same or different, and may further containheteroatoms. Preferred chemical linkers comprise a backbone of one oremore ethyleneglycol or beta-amino acid, or gamma-amino acid moieties.Also cyclic linkers may be used such as. Examples for cyclic linkersinclude without limitation 3-amino-4-pyrazolecarboxylic acid,4-amino-5-carboxy-2-hydroxypyrimidine, anthranilic acid derivatives,3-amino-1-indanecarboxylic acid.

In accordance with a preferred embodiment of the above aspects, thepolypeptide has a length of 9, 10, 11, 12, 13, or 14 amino acidresidues. More preferred is a length of 9 or 10 amino acid residues.

In accordance with another preferred embodiment of the above aspects,said polypeptides consist of said amino acids.

Reference to anti-angiogenic activity is determined by assays hereindisclosed. For example, the polypeptides of the invention are tested byin vitro assays which include the inhibition of endothelial cellmediated tubule formation, inhibition of endothelial cell migration,inhibition of VEGF and bFGF induced endothelial cell proliferation andendothelial cell cytotoxicity assays. In a preferred embodiment, theanti-angiogenic compounds of the present invention inhibit tubuleformation, cell migration or cell proliferation by at least 10 percent,preferably at least 20 percent, more preferably at least 30 percent andmost preferably at least 50 percent relative to a control without saidpolypeptide in an assay as herein described. In a more preferredembodiment, the assay is a proliferation assay according to the Examplehereinbelow.

Polypeptides can also be tested in vivo using murine tumour models asherein disclosed. In a preferred embodiment, the anti-angiogeniccompounds of the present invention inhibit tumour growth by at least 10percent, preferably at least 20 percent, more preferably at least 30percent and most preferably at least 50 percent relative relative to acontrol without said polypeptide.

A variant, i.e. a fragment polypeptide and reference polypeptide maydiffer in amino acid sequence by one or more substitutions, additions,deletions, truncations which may be present in any combination. Amongpreferred variants are those that vary from a reference polypeptide byconservative amino acid substitutions. Such substitutions are those thatsubstitute a given amino acid by another amino acid of likecharacteristics. The following non-limiting list of amino acids areconsidered conservative replacements (similar): a) serine, andthreonine; b) glutamic acid and asparatic acid; c) asparagine andglutamine d) arginine and lysine; e) isoleucine, leucine, methionine andvaline and f) phenylalanine, histidine, tyrosine and tryptophan. Mosthighly preferred are variants which retain the same biological functionand activity as the reference polypeptide from which it varies.

A functionally equivalent polypeptide according to the invention is avariant wherein one or more amino acid residues are substituted withconserved or non-conserved amino acid residues, or one in which one ormore amino acid residues includes a substituenti group. Conservativesubstitutions are the replacements, one for another, among the aliphaticamino acids Ala, Val, Leu and Ile; interchange of the hydroxl residuesSer and Thr; exchange of the acidic residues Asp and Glu; substitutionbetween amide residues Asn and Gln; exchange of the basic residues Lysand Arg; and replacements among aromatic residues Phe, His, Tyr and Trp.

In addition, the invention features polypeptide sequences having atleast 75% identity with the polypeptide sequences as herein disclosed,or fragments and functionally equivalent polypeptides thereof. In oneembodiment, the polypeptides have at least 85% identity, more preferablyat least 90% identity, even more preferably at least 95% identity, stillmore preferably at least 97% identity, and most preferably at least 99%identity with the amino acid sequences illustrated herein.

The polypeptides of the present invention are preferably composed ofalpha-amino acid residues. The invention, however, is not limited topolypeptides composed of alpha-amino acid residues, polypeptidescomprising other types of residues, such as beta- or gamma-amino acidresidues are also contemplated by the present invention. Any type ofsuitable residues may be used in accordance with the present inventionso long as the polypeptide does not lose its ability to adopt therequired biologically active three dimensional conformation.

According to one aspect to the invention there is provided apolypeptide, or part thereof, comprising an amino acid sequence asrepresented by the sequence presented in FIG. 1 which has been modifiedby deletion, addition or substitution of at least one amino acid residuewherein said modified polypeptide has anti-angiogenic activity.

According to another aspect of the invention there is provided apolypeptide comprising an amino acid sequence selected from the groupconsisting of:

i) a peptide of the sequence, or part thereof: XXXXXLXEXXGXXXPRVXXRwherein X is any amino acid residue;

-   ii) a peptide as represented in (i) wherein amino acid residue X is    selected from the following group: alanine, valine, leucine,    isoleucine, proline; and-   iii) a peptide represented in (i) or (ii) which has anti-angiogenic    activity.

In a preferred embodiment of the invention said polypeptide comprises anamino acid sequence, or part thereof, consisting of the sequence:SXXXXXLXEXXGXXXPRVXXR

In a preferred embodiment of the invention said polypeptide comprises anamino acid sequence as represented by the sequence: XXXXXLXEXXGXXXPRVVXR

In a yet further preferred embodiment of the invention said polypeptidecomprises an amino acid sequence as represented by the sequence:GEGXFLXEXXGXXXPRVVXR

In a yet further preferred embodiment of the invention said polypeptidecomprises an amino acid sequence as represented by the sequence: GEG XFLXXX XXXXX XXXX XX.

In a preferred embodiment of the invention said polypeptide comprises anamino acid sequence as represented by the sequences presented intable 1. Preferably said polypeptides comprising said sequence haveanti-angiogenic activity.

In a yet further preferred embodiment of the invention said polypeptidecomprises an amino acid sequence selected from the group consisting of:GEG DFL AEG GGV RGP RVVE R GEG DFL AEG GGX RGP RVVE R GEG DFL AEG GGVXGP RVVE R GEG DFL AEG GGV RXP RVVE R GEG DFL AEG GGV RGP RVXE R GEG DFLAEG GGV RGP RVVXR GEG DFL AEG GGXXXP RVVX R GEG DFL AEG GGXXXP RVXXR.

X is any amino acid residue or, preferably, X is selected from the groupconsisting of alanine, valine, leucine, isoleucine, or proline. Morepreferably X is alanine.

In yet a further preferred embodiment of the invention the peptidecomprises an amino acid sequence as represented by the overlapping partof two fragments presented in table 1. In a more preferred embodiment,said peptide is derived from the overlapping part of the peptidesAHI-401 and AHI-378 in table 1. Preferably, said peptide derived fromthe overlapping part of the peptides AHI-401 and AHI-378 comprises oneadditional amino acid residue at the N-terminus.

Thus, in a preferred method of the invention said peptide comprises anamino acid sequence as represented by the sequence: XFLAEGGGVXG

X is any amino acid residue selected from the group consisting of A, R,N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V. Preferably X isselected from the group consisting of A, V, L, I and P, more preferablyX is a basic amino acid selected from the group consisting of H, R andK, or an acidic amino acid selected from the group consisting of D andE.

In another preferred embodiment, the N-terminal X is selected from thegroup consisting of D and E, whereas the C-terminal X is selected fromthe group consisting of H, R and K, or alternatively, the N-terminalamino acid is selected from the group consisting of H, R and K, whereasthe C-terminal X is selected from the group consisting of D and E. In aparticularly preferred embodiment, the N-terminal X is D and theC-terminal X is R.

In another preferred embodiment, the C-terminal X is selected from thegroup consisting of L-2,4-diaminobutyric acid, R-aminocarnitine,L-alpha-amino-gamma-guanidinobutyric acid or ornithine.

In a further preferred embodiment of the invention said part thereof isrepresented by the amino acid sequence from+1 to+15 of the amino acidsequence: SXXXXXLXEXXGXXXPRVXXR

In a further preferred embodiment of the invention said part thereof isrepresented by the amino acid sequence from+6 to+21 of the amino acidsequence: XXXXXLXEXXGXXXPRVXXR

In a yet further preferred embodiment of the invention said part thereofis represented by the amino acid sequence+6 to+15 of the amino acidsequence XXXXXLXEXXGXXXPRVXXR

In a further preferred embodiment of the invention said polypeptideconsists of the peptide amino acid sequences as herein disclosed.

In a further preferred embodiment of the invention said polypeptide is10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 amino acidresidues in length and comprises an amino acid sequence according to theinvention.

It will be apparent to one skilled in the art that modification to theamino acid sequence of polypeptides according to the invention couldenhance the binding and/or stability of the polypeptide with respect toits target sequence. In addition, modification of the polypeptide mayalso increase pharmacological properties of the polypeptide, such as forexample the in vivo stability, thereby reducing the effective amount ofpolypeptide necessary to inhibit angiogenesis. This would advantageouslyreduce undesirable side effects which may result in vivo.

Numerous peptide-modifications suitable for enhancing thepharmacological properties of the polypetide are known in the art. Forexample, the peptide backbone can be chemically modified, for instanceas described in Gilles Guichard, Solid-phase synthesis of pseudopeptidesand oligomeric peptide backbone mimetics, in Solid-phase synthesis—Apractical guide,; edited by Steven A. Kates or in Fernando Albericio,Marcell Dekker, New York—Basel, pp 649-703, 2000, the contents of whichare herewith incorporated by reference. Examples of backbonemodifications include without limitation: beta-peptides, depsipeptides,oligosulfonamides, oliogureas and thioureas, oligocarbamates, peptoids,and azapeptides.

Another example of a suitable modification is the cyclisation of apeptide according to the invention. Said cyclisation may be head to tailcyclisation, side-chain to side-chain cyclisation, side-chain to endcyclisation, branched cyclisation, or backbone to backbone cyclisation.Cyclisation of peptides are for instance described in the followingreferences, the contents of which are herewith incorporated byreference: Paolo Rovero, Homodetic cyclic peptides, in Solid-phasesynthesis—A practical guide,; edited by Steven A. Kates and FernandoAlbericio, Marcell Dekker, New York—Basel, pp 331-364, 2000. S A Kates,N A Sole, F Albericio, G. Barany, in C. Basava, G M Anantharamaiah, eds.Peptides: design, synthesis, and biological activity. Boston,Birkhauser, 1994, pp 39-58.

Other suitable modifications include the incorporation of “non-proteinamino acids”.

As used herein, the term “amino acid” or “proteinogenic amino acid”refer to an amino acid which is a natural building block of a protein,i.e. an amino acid which is incorporated into a protein in the processof translating the genetic code into a protein. The interchangeably usedterms “non-protein amino acid”, “non-proteinogenic amino acid” or“non-coded amino acid” include any compound with a chemical structuresimilar to an amino acid, but not naturally used in the process oftranslating the genetic code into a protein.

Accordingly, proteinogenic amino acids may for instance be replaced bythe alpha-methyl derivative or by the alpha-ethyl derivative. In apreferred embodiment the peptide comprises an alphamethyl derivativeselected from the group consisting of: alpha-methyl-L-alanine,alpha-methyl-L-arginine, alpha-methyl-L-asparagine,alpha-methyl-L-aspartic acid, alpha-methyl-Lcysteine,alpha-methyl-L-glutamatic acid, alpha-methyl-L-glutamine,alpha-methyl-L-glycine, alpha-methyl-L-histidine,alpha-methyl-L-isoleucine, alpha-methyl-L-leucine,alpha-methyl-L-lysine, alpha-methyl-L-methionine,alpha-methyl-L-phenylalanine, alpha-methyl-L-proline,alpha-methyl-L-serine, alpha-methyl-1-threonine,alpha-methyl-L-tryptophan, alpha-methyl-L-tyrosine andalpha-methyl-L-valine.

Another example of a suitable modification may be the replacement ofamino acids for intstance by the N-methyl derivative or the N-ethylderivative. In another preferred embodiment the peptide comprises anN-methyl derivative selected from the group consisting of:N-methyl-L-alanine, N-methyl-L-arginine, N-methyl-L-asparagine,N-methyl-L-aspartic acid, N-methyl-L-cysteine, N-methyl-L-glutamic acid,N-methyl-L-glutamine, N-methyl-L-glycine, N-methyl-L-histidine,N-methyl-L-isoleucine, N-methyl-L-leucine, N-methyl-L-lysine,N-methyl-L-methionine, N-methyl-L-phenylalanine, N-methyl-L-proline,N-methyl-L-serine, N-methyl-L-threonine, N-methyl-L-tryptophan,N-methyl-L-tyrosine and N-methyl-L-valine.

As will be apparent to the person of skill in the art, groups of aminoacids with similar properties (for instance hydrophobic amino acids) arepreferably replaced by “non-protein” amino acids with similarproperties, analogous to what is known in the art as conservative aminoacid substitutions. As used herein, “conservative” amino acids includeboth proteinogenic and non-proteinogenic amino acids with similarproperties. Thus, a hydrophobic proteinogenic amino acid may beexchanged by a hydrophobic non-proteinogenic amino acid, a polarproteinogenic amino acids may be exchanged by a polar non-proteinogenicamino acids etc.

Accordingly, hydrophobic amino acid may be replaced byalpha,alpha-disubstituted linear or cylic amino acid. Without intendingto be bound to these examples, hydrophobic amino acids such as glycine,alanine, leucine, isoleucine, valine, phenylalanine or proline may bereplaced by 1-aminocyclopropanecarboxylic acid,3-aminopentane-3-carboxylic acid, R-2-amino-2-cyclohexyl-propanoic acid;acidic amino acids such as glutamic acid or aspartic acid may bereplaced by gamma-carboxy-L-glutamic acid, L-2-aminoadipic acid,L-3-aminoadipic acid or (+/−)-lminocyclopentane-cis-1,3-dicarboxylicacid acid. Further examples include, Cha, O-NaI, Aib (aminoisobutyricacid), Ac_(n)c (α,α-disubstituted cyclic α-amino acid, n from 3 to 7,both inclusived; n refers to the number of carbons in the ring), Abu(2-aminobutyric acid), Nle, Nva (norvaline), Bpa(p-benzoyl-phenylalanine), hphe (homo-Phe), hPro (homo-Pro),1-Nal(p-(1-naphthyl)alanine), 2-Nal(3-(2-naphthyl)alanine), Oic(octahydroindode-2-carboxylic acid), Tic(1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid), Pen (penicillamine),Phg (phenylglycine), Tle (tert-leucine), p-X-Phe (X=Br, F, I, Cl,phenyl, CN, NO₂), Thi α-(2-thienyl)-alanine), and their homologues.

Furthermore, single proteinogenic amino acids may also be replaced bystructurally similar “non-protein” amino acids derivatives. Structurallysimilar derivatives for a given proteinogenic amino acid are known inthe art. For instance, arginine may be replaced by L-2,4-diaminobutyricacid, R-aminocarnitine, L-alpha-amino-gamma-guanidinobutyric acid orornithine; phenylalanine may be replaced by L-2-naphthylalanine,L-1,2,34-tetrahydroisoquinoline-3-carboxylic acid or4,4-biphenylalanine, p-benzoyl-phenylalanine, hphe; proline by3-hydroxyproline, 4-hydroxyproline; lysine by 5-hydroxylysine,allo-hydroxylysine, N⁶-acetyllysine, N⁶-methyllysine,N⁶,N⁶-dimethyllysine, N⁶,N⁶,-trimethyllysine; alanine bycyclohexyalanine. Other modifications include amino acids with a C₂, C₃or C₄ alkyl R group optionally substituted by 1, 2 or 3 substituentsselected from halo (eg F, Br, I), hydroxy or C₁-C₄ alkoxy.

Another suitable modification of the peptides of the invention is thereplacement of one or more L-amino acids may by the correspondingD-amino acid. Both, the proteinogenic or “non-protein” L-amino acids maybe replaced by the corresponding D-amino acids.

A further suitable peptide derivative in accordance with the presentinvention is a retro-inverso peptidomimetic as described in thefollowing references, the content of which herewith is incorporated byreference: M. Goodman, M. Chorev Acc Chem Res 12, 1-7, 1979, M. Chorev,M. Goodman Acc Chem Res 26, 266-273, 1993 and M. Chorev, M. GoodmanTrend Biotechnol 13, 438-445, 1995.

In another embodiment of the present invention, the C-terminus ismodified. C-terminal modifications include, by examples and not by wayof limitation: carboxylic acid, esters, carboxamides, or alcohol. In apreferred embodiment, the C-terminal modification is a methyl-, ethyl-,propyl-, butyl- or isopropyl-ester. In another preferred embodiment, theC-terminal modification is CONH₂, CONHCH₃ (methyl amide) or CON(CH₃)₂(dimethylamide). In another preferred embodiment, the C-terminalmodification is CH₂OH.

In further embodiment of the present invention, the N-terminus ismodified. N-terminal modifications include, by example and not by way oflimitation, carboxamide, carbamate, urea, sulphonamide, acetylation andalkylation.

In a further preferred embodiment of the invention said polypeptide, ismodified at both C-terminus and N-terminus.

Alternatively or preferably, said modification includes the use ofmodified amino acids in the production of recombinant or synthetic formsof polypeptides according to the invention.

In a further preferred embodiment of the invention there is provided apolypeptide according to the invention which polypeptide comprises atleast one modified amino acid wherein X denotes the position of saidmodified amino acid.

The incorporation of modified amino acids may confer advantageousproperties on polypeptides according to the invention. For example, theincorporation of modified amino acids may increase the affinity of thepolypeptide for its binding site, or the modified amino acids may conferincreased in vivo stability on the polypeptide thus allowing a decreasein the effective amount of therapeutic polypeptide administered to apatient.

It will also be apparent to one skilled in the art that fragments ofpolypeptides which retain anti-angiogenic activity could be recovered byfractionation of the intact polypeptide using, for example, proteolyticenzymes. Alternatively, fragments could be synthesised de novo and alsomodified by, for example, cyclisation. Cyclisation is known in the art,(see Scott et al Chem Biol (2001), 8:801-815; Gellerman et al J. PeptideRes (2001), 57: 277-291; Dutta et al J. Peptide Res (2000), 8:398-412;Ngoka and Gross J Amer Soc Mass Spec (1999), 10:360-363.

In a preferred embodiment of the invention the polypeptides according tothe invention are modified by cyclisation.

According to a further aspect the invention there is provided apharmaceutical composition comprising a polypeptide, or part thereof,according to the invention.

According to a further aspect of the invention there is provided anpharmaceutical composition comprising two or more polypeptides accordingto the invention wherein said pharmaceutical composition hasanti-angiogenic activity.

In a preferred embodiment of the invention said two or more polypeptidesare linked by a linker molecule. Preferably said linker molecule is aflexible linker.

In a further preferred embodiment of the invention said pharmaceuticalcomposition comprises a plurality of polypeptides according to theinvention. Preferably said pharmaceutical composition has 3, 4, 5, 6, 7,8, 9, or 10 polypeptides linked together as an oligomeric polypeptide.Preferably said polypeptide has greater than 10 polypeptides accordingto the invention.

In a further preferred embodiment of the invention said pharmaceuticalcomposition is a dimer of two polypeptides according to the invention.

In a further preferred embodiment of the invention said linker is apolypeptide linking molecule. In a preferred embodiment of the inventionsaid polypeptide linking molecule comprises at least one amino acidresidue which links at least two polypeptides according to theinvention.

In a further preferred embodiment of the invention said polypeptidelinking molecule comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 aminoacid residues. In a further embodiment of the invention said linkingmolecule comprises more than 10 amino acid residues.

In an alternative embodiment of the invention, the polypeptide is afusion protein comprising an inframe translational fusion of thepolypeptides according to the invention.

It will be apparent that the invention encompasses the formationoligomeric polypeptides which comprise identical polypeptides accordingto the invention, herein referred to as homo-oligomeric as well aspolypeptides comprising different modified polypeptides, referred to ashetero-oligomeric.

It will be apparent to one skilled in the art that alternative linkerscan be used to link polypeptides, for example the use of chemicalprotein crosslinkers. For example homo-bifunctional crosslinker such asdisuccinimidyl-suberimidate-dihydrochloride;dimethyl-adipimidate-dihydrochloride; 1,5,-2,4 dinitrobenezene orhetero-bifunctional crosslinkers such as N-hydroxysuccinimidyl2,3-dibromopropionate; 1-ethyl-3-[3-dimethylaminopropyl] carbodiimidehydrochloride; succinimidyl4-[n-maleimidomethyl]-cyclohexane-1-carboxylate.

When administered, the pharmaceutical compositions of the presentinvention are administered in pharmaceutically acceptable preparations.Such preparations may routinely contain pharmaceutically acceptableconcentrations of salt, buffering agents, preservatives, compatiblecarriers, supplementary immune potentiating agents such as adjuvants andcytokines and optionally other therapeutic agents, such aschemotherapeutic agents.

The pharmaceutical compositions of the invention can be administered byany conventional route, including injection or by gradual infusion overtime. The administration may, for example, be oral, intravenous,intraperitoneal, intramuscular, intracavity, subcutaneous, ortransdermal.

The compositions of the invention are administered in effective amounts.An “effective amount” is that amount of a composition that alone, ortogether with further doses, produces the desired response. In the caseof treating a particular disease, such as cancer, the desired responseis inhibiting the progression of the disease. This may involve slowingthe progression of the disease temporarily, although more preferably, itinvolves halting the progression of the disease permanently. This can bemonitored by routine methods.

Such amounts will depend, of course, on the particular condition beingtreated, the severity of the condition, the individual patientparameters including age, physical condition, size and weight, theduration of the treatment, the nature of concurrent therapy (if any),the specific route of administration and like factors within theknowledge and expertise of the health practitioner. These factors arewell known to those of ordinary skill in the art and can be addressedwith no more than routine experimentation.

The pharmaceutical compositions used in the foregoing methods oftreatment preferably are sterile and contain an effective amount ofpolypeptide, oligomeric agent or nucleic acid encoding said polypeptideor oligomeric agent, for producing the desired response in a unit ofweight or volume suitable for administration to a patient.

The doses of polypeptide/oligomer, or nucleic acid encoding saidpolypeptide/oligomer administered to a subject can be chosen inaccordance with different parameters, in particular in accordance withthe mode of administration used and the state of the subject. Otherfactors include the desired period of treatment. In the event that aresponse in a subject is insufficient at the initial doses applied,higher doses (or effectively higher doses by a different, more localizeddelivery route) may be employed to the extent that patient tolerancepermits.

When administered, the therapeutic preparations of the invention areapplied in therapeutically-acceptable amounts and inpharmaceutically-acceptable compositions. The term “pharmaceuticallyacceptable” means a non-toxic material that does not interfere with theeffectiveness of the biological activity of the active ingredients. Suchpreparations may routinely contain salts, buffering agents,preservatives, compatible carriers, and optionally other therapeuticagents. When used in medicine, the salts should be pharmaceuticallyacceptable, but non-pharmaceutically acceptable salts may convenientlybe used to prepare pharmaceutically-acceptable salts thereof and are notexcluded from the scope of the invention.

Polypeptide/oligomer polypeptide compositions may be combined, ifdesired, with a pharmaceutically-acceptable carrier. The term“pharmaceutically-acceptable carrier” as used herein means one or morecompatible solid or liquid fillers, diluents or encapsulating substanceswhich are suitable for administration into a human. The term “carrier”denotes an organic or inorganic ingredient, natural or synthetic, withwhich the active ingredient is combined to facilitate the application.

The pharmaceutical compositions may contain suitable buffering agents,including: acetic acid in a salt; citric acid in a salt; boric acid in asalt; and phosphoric acid in a salt.

The pharmaceutical compositions also may contain, optionally, suitablepreservatives, such as: benzalkonium chloride; chlorobutanol; parabensand thimerosal.

The pharmaceutical compositions may conveniently be presented in unitdosage form and may be prepared by any of the methods well-known in theart of pharmacy. All methods include the step of bringing the activeagent into association with a carrier which constitutes one or moreaccessory ingredients. In general, the compositions are prepared byuniformly and intimately bringing the active compound into associationwith a liquid carrier, a finely divided solid carrier, or both, andthen, if necessary, shaping the product.

Compositions suitable for oral administration may be presented asdiscrete units, such as capsules, tablets, lozenges, each containing apredetermined amount of the active compound. Other compositions includesuspensions in aqueous liquids or non-aqueous liquids such as a syrup,elixir or an emulsion.

Compositions suitable for parenteral administration convenientlycomprise a sterile aqueous or non-aqueous preparation ofpolypeptides/oligomer or nucleic acids, which is preferably isotonicwith the blood of the recipient. This preparation may be formulatedaccording to known methods using suitable dispersing or wetting agentsand suspending agents. The sterile injectable preparation also may be asterile injectable solution or suspension in a non-toxicparenterally-acceptable diluent or solvent, for example, as a solutionin 1,3-butane diol. Among the acceptable vehicles and solvents that maybe employed are water, Ringer's solution, and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose any bland fixed oilmay be employed including synthetic mono-or di-glycerides. In addition,fatty acids such as oleic acid may be used in the preparation ofinjectables.

Carrier formulation suitable for oral, subcutaneous, intravenous,intramuscular, etc. administrations can be found in Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa.

In a preferred embodiment of the invention said pharmaceuticalcompositions modulates angiogenesis. Preferably said modulation is theinhibition of angiogenesis.

Preferably said inhibition relates to endothelial cell stimulatedangiogenesis.

Alternatively, or preferably, said inhibition is the inhibition ofmacrophage and/or tumour cell stimulated angiogenesis.

In a further preferred embodiment of the invention said inhibition ismediated by the inhibition of pro-angiogenic factors. Ideally these areeither intracellular or cell surface receptors.

More preferably still, said inhibition is mediated via inhibition of theactivity of pro-angiogenic growth factors. Ideally said growth factorsare selected from: VEGF, bFGF; aFGF; TGFβ; PDGF.

According to a yet further aspect of the invention there is provided theuse of a polypeptide according to the invention, or part thereof, and/oroligomers for the manufacture of a medicament for use in the treatmentof cancer.

According to a further aspect of the invention there is provided anucleic acid molecule comprising DNA sequences selected from:

-   i) the DNA sequence as represented in FIG. 2;-   ii) the DNA sequence as represented in FIG. 2 which has been    modified by addition, deletion, or substitution of at least one    nucleotide base within at least one codon to encode a modified    peptide according to the invention;-   iii) DNA sequences which hybridise to the sequences presented in    FIG. 2 which encode a peptide having anti-angiogenic activity; and-   iv) DNA sequences which are degenerate as a result of the genetic    code to the DNA sequences defined in (i), (ii) or (iii).

In a preferred embodiment of the invention there is provided an isolatednucleic acid molecule which anneals under stringent hybridisationconditions to the sequences described in (i), (ii), (iii) and (iv)above.

Stringent hybridisation/washing conditions are well known in the art.For example, nucleic acid hybrids that are stable after washing in0.1×SSC, 0.1% SDS at 60° C. It is well known in the art that optimalhybridisation conditions can be calculated if the sequence of thenucleic acid is known. Typically, hybridisation conditions uses 4-6×SSPE(20×SSPE contains 175.3 g NaCl, 88.2 g NaH₂PO₄H₂O and 7.4 g EDTAdissolved to 1 litre and the pH adjusted to 7.4); 5-10× Denhardtssolution (50× Denhardts solution contains 5 g Ficoll (Type 400,Pharmacia), 5 g polyvinylpyrrolidone abd 5 g bovine serum albumen; 1001g-1.0 mg/ml sonicated salmon/herring DNA; 0.1-1.0% sodium dodecylsulphate; optionally 40-60% deionised formamide. Hybridisationtemperature will vary depending on the GC content of the nucleic acidtarget sequence but will typically be between 420-65° C.

The present invention further provides, in another preferred embodiment,an isolated nucleic acid molecule consisting of a DNA sequence encodingone or more of the peptide amino acid sequences as disclosed herein.

Polypeptides according to the invention can be manufactured by in vitropeptide synthesis using standard peptide synthesis techniques.Alternatively, or preferably, polypeptides can be manufactured byrecombinant techniques which are well known in the art.

According to a further aspect of the invention there is provided avector, wherein said vector includes a nucleic acid molecule whichencodes for polypeptides and/or oligomers according to the invention. Ina preferred embodiment, the vector encodes one or more of the peptideamino acid sequences as disclosed herein.

Alternatively, vector(s) which include nucleic acid encoding saidpolypeptides can be adapted for recombinant expression.

In a preferred embodiment of the invention said vector is an expressionvector adapted for prokaryotic or eukaryotic cell expression. Preferablysaid eukaryotic vector is adapted for gene therapy.

Typically said adaptation includes, by example and not by way oflimitation, the provision of transcription control sequences (promotersequences) which mediate cell/tissue specific expression. These promotersequences may be cell/tissue specific, inducible or constitutive.

Promoter is an art recognised term and, for the sake of clarity,includes the following features which are provided by example only, andnot by way of limitation. Enhancer elements are cis acting nucleic acidsequences often found 5′ to the transcription initiation site of a gene(enhancers can also be found 3′ to a gene sequence or even located inintronic sequences and is therefore position independent). Enhancersfunction to increase the rate of transcription of the gene to which theenhancer is linked. Enhancer activity is responsive to trans actingtranscription factors (polypeptides) which have been shown to bindspecifically to enhancer elements. The binding/activity of transcriptionfactors (please see Eukaryotic Transcription Factors, by David SLatchman, Academic Press Ltd, San Diego) is responsive to a number ofenvironmental cues which include, by example and not by way oflimitation, intermediary metabolites or environmental effectors.

Promoter elements also include so called TATA box and RNA polymeraseinitiation selection (RIS) sequences which function to select a site oftranscription initiation.

These sequences also bind polypeptides which function, inter alia, tofacilitate transcription initiation selection by RNA polymerase.

Adaptations also include the provision of selectable markers andautonomous replication sequences which both facilitate the maintenanceof said vector in either the eukaryotic cell or prokaryotic host.Vectors which are maintained autonomously are referred to as episomalvectors.

Adaptations which facilitate the expression of vector encoded genesinclude the provision of transcription termination/polyadenylationsequences. This also includes the provision of internal ribosome entrysites (IRES) which function to maximise expression of vector encodedgenes arranged in bicistronic or multi-cistronic expression cassettes.

These adaptations are well known in the art. There is a significantamount of published literature with respect to expression vectorconstruction and recombinant DNA techniques in general. Please see,Sambrook et al (1989) Molecular Cloning: A Laboratory Manual, ColdSpring Harbour Laboratory, Cold Spring Harbour, N.Y. and referencestherein; Marston, F (1987) DNA Cloning Techniques: A Practical ApproachVol m IRL Press, Oxford UK; DNA Cloning: F M Ausubel et al, CurrentProtocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

In a yet further preferred embodiment of the invention there is provideda gene therapy vector comprising the nucleic acid according to theinvention.

It will be apparent to one skilled in the art that the delivery of genetherapy vectors either to endothelial cells or tumour cells target theproduction of polypeptides according to the invention to the vicinity ofthe tumour thereby augmenting the anti-angiogenic effect of saidpolypeptides.

According to a yet further aspect of the invention there is provided acell transformed/transfected with the nucleic acid according to theinvention. Ideally said nucleic acid is the vector according to theinvention.

According to a further aspect of the invention there is provided amethod for the production of polypeptides according to the inventioncomprising:

-   i) providing a cell according to the invention;-   ii) providing conditions conducive to the manufacture of    polypeptides according to the invention; and-   iii) purifying said polypeptides from a cell, or a cells culture    environment.

According to yet still a further aspect of the invention there isprovided a non-human, transgenic animal characterised in that saidanimal incorporates a nucleic acid molecule encoding a polypeptideaccording to the invention into its genome.

It will be apparent to one skilled in the art that the provision ofnon-human transgenic animals genetically modified by the provision of atransgene(s) encoding polypeptides according to the invention is analternative source of active polypeptide. It is well known in the artthat transgenic animals can be used to make various therapeuticpolypeptides.

In a preferred embodiment of the invention said transgene is of humanorigin.

In a further aspect of the invention there is provided a method to treatan animal which would benefit from inhibition of angiogenesiscomprising:

-   i) administering an effective amount of an agent comprising a    polypeptide according to the invention, to an animal to be treated;-   ii). monitoring the effects of said agent on the inhibition of    angiogenesis.

In a preferred method of the invention said treatment is the inhibitionof tumour development.

In an alternative method of treatment, polypeptides according to theinvention are additionally conjugated, associated or crosslinked to anagent which augments the anti-angiogenic effect of thepolypeptide/oligomer.

Typically the agent could be a cytotoxic agent, another anti-angiogenicagent, a prodrug activating enzyme, a chemotherapeutic agent, apro-coagulant agent or immunomodulatory factor.

Examples of these are well known in the art, for example, and not by wayof limitation cytotoxins, such as ricin A-chain or diphtheria toxin;antagonists of the key pro-angiogenic factors in tumours (eg VEGF, bFGF,TNF alpha, PDGF) would include neutralising antibodies or receptors forthese factors, or tyrosine kinase inhibitors for their receptors (eg.PTK787 for the VEGF receptor, Flk-1/KDR); prodrug activating enzymessuch as, human simplex virus-thymidine kinase HSV-TK, which activatesthe prodrug, ganciclovir when it is then admininistered systemically;chemotherapeutic agents, such as neocarzinostatin; cisplatin;carboplatin; cyclosphosphamide; melphalan; carmusline; methotrexate;5-fluorouracil; cytarabine; mercaptopurine; daunorubicin; doxorubicin;epirubicin; vinblastine; vincristine; dactinomycin; mitomycin C; taxol;L-asparaginase; G-CSF; an enediyne such as chalicheamicin oresperamicin; chlorambucil; ARA-C; vindesine; bleomycin; and etoposide.

In addition, or alternatively, the cell surface domain of human tissuefactor (this truncated form of tissue factor (tTF) could also beassociated with polypeptides according to the invention. Truncated TFhas limited anti-endothelial activity when free in the circulation, butbecomes an effective and selective thrombogen (eg it causes extensivethrombosis and coagulation in blood vessels) when targeted to thesurface of tumor endothelial cells.

An example of an immunomodulatory factor is the Fc effector domain ofhuman IgG1. This binds natural killer (NK) cells and also the C1qprotein that initiates the complement cascade. NK cells and complementthen activate a powerful cytolytic response against the targetedendothelial cells.

It will be apparent that the above combinations of polypeptides andtherapeutic agents will also have benefit with respect to the treatmentof other conditions/diseases which are dependent on angiogenesis. Forexample, neovascular glaucoma, rheumatoid arthritis, psoriasis anddiabetic retinopathy.

In a yet further alternative method of treatment, said gene therapyvector includes, and therefore said nucleic acid encoding a polypeptideaccording to the invention is provided with, nucleic acid encoding anagent which augments the anti-angiogenic effect of said polypeptide.

According to a yet further aspect of the invention there is provided animaging agent comprising a polypeptide according to the invention.Examples of suitable imaging agents include, but are not limited to,contrast agents, magnetic resonance imaging agents, a paramagneticisotope, a heavy metal, or a radioisotope.

It will be apparent to the skilled artisan that polypeptides accordingto the invention can be used to target imaging agents to, for example,tumours, to identify developing tumours or to monitor the effects oftreatments to inhibit tumour growth. It will also be apparent that thecombined therapeutic compositions which comprise both polypeptides and afurther anti-angiogenic agent may be further associated with an imagingagent to monitor the distribution of the combined therapeuticcomposition and/or to monitor the efficacy of said combined composition.

Methods used to detect imaging agents are well known in the art andinclude, by example and not by way of limitation, positron emissiontomographic detection of F¹⁸ and C¹¹ compounds.

According to a further aspect of the invention there is provided ascreening method for the identification of ligands which modulate theinteraction of a polypeptide/oligomer according to the invention with avitronectin receptor.

In a preferred method of the invention said method comprises the stepsof:

-   i) providing a polypeptide comprising the amino acid sequence    presented in FIG. 3 or active binding fragment thereof;-   ii) providing at least one polypeptide or agent comprising an amino    acid sequence selected from the sequences disclosed herein;-   iii) providing at least one ligand to be tested;-   iv) forming a preparation of (i), (ii) and (iii); and-   v) detecting or measuring the effect of the ligand in (iii) on the    interaction of the polypeptide in (i) with the polypeptide or agent    in (ii).

In a further preferred method of the invention said ligand ispre-incubated with polypeptide in (i) prior to addition of thepolypeptide or agent in (ii).

In an alternative preferred method of the invention said ligand ispre-incubated with the polypeptide or agent in (ii) prior to addition tothe polypeptide in (i).

In a further preferred method of the invention said vitronectin receptoris presented in a soluble form or by a cell. Preferably said cellnaturally expresses the vitronectin receptor. Examples of cells whichnaturally express the vitronectin receptor are endothelial cells, smoothmuscle cells, osteoclasts and tumour cells. Alternatively said cell doesnot naturally express the vitronectin receptor, in which case said cellsare preferably genetically engineered to express the vitronectinreceptor.

According to a further aspect of the invention there are providedagent(s) identified by the screening method according to the invention.Preferably said agent(s) interfere with the interaction ofpolypeptides/oligomers with vitronectin receptor binding activity.Alternatively said agent promotes the interaction of polypeptides withvitronectin receptor binding activity.

An embodiment of the invention will now be described, by example only,and with reference to the following figures:

FIG. 1 represents the amino acid sequence of α1-24;

FIG. 2 represents the nucleic acid sequence encoding α1-24;

FIG. 3 represents a summary of the anti-angiogenic activity ofpolypeptides as herein disclosed.

FIGS. 4 and 5 show tubule formation activity of peptides according tothis invention.

Materials and Methods

Adult human dermal microvascular endothelial cells (HuDMECs) wereobtained commercially (TCS Biologicals, Buckinghamshire, United Kingdom)and cultured in microvascular endothelial cell growth medium (EGM). Thismedium contains heparin (10 ng/ml), hydrocortisone, human epidermalgrowth factor (10 ng/ml), human fibroblast growth factor (10 ng/ml)(such endothelial growth factors are necessary for routine passaging ofHuDMECs in culture) and dibutyryl cyclic AMP. This was supplemented with5% heat-inactivated FCS, 50% g/ml gentamicin and 50 ng/ml amphotericin B(TCS Biologicals, United Kingdom). Murine endothelial cells (SVEC 4-10)were obtained from the ATCC and cultured in DMEM+10% FCS. Cells weregrown at 37° C. in a 100% humidified incubator with a gas phase of 5%CO₂ and routinely screened for Mycoplasma. Prior to their use in theassays indicated below, HuDMECs were grown to 80% confluency, incubatedin DMEM+1% FCS for 2h, then harvested with 0.05% trypsin solution,washed twice and resuspended to the cell density required for each assay(see below).

Peptide Synthesis and Analysis

Experimental Conditions for Analytical HPLC

Gradient 1: linear gradient over 2.5 min of MeCN/0.09% TFA and H₂O/0.1%TFA from 1:49 to 3:2; flow rate 4.0 mL/min, detection at 215 nm; SB-C18ZORBAX, column 3.5 μm, 50×4.6 mm.

Gradient 2: linear gradient over 2.5 min of MeCN/0.09% TFA and H₂O/0.1%TFA from 1:49 to 1:0; flow rate 4.0 mL/min, detection at 215 nm; SB-C18ZORBAX column, 3.5 μm, 50×4.6 mm.

Gradient 3: linear gradient over 2.5 min of MeCN/0.09% TFA and H₂O/0.1%TFA from 1:49 to 3:2; flow rate 4.0 mL/min, detection at 215 nm;Nucleosil C8 column, 5.0 μm, 125×4.0 mm.

Gradient 4: linear gradient from over 2.5 min of MeCN/0.09% TFA andH₂O/0.1% TFA from 1:49 to 3:2; flow rate 4 ml/min; detection at 215 nm;Chromolith SpeedROD C₁₈ column, 50×4.6 mm.

Gradient 5: linear gradient from over 2.5 min of MeCN/0.09% TFA andH₂O/0.1% TFA from 1:49 to 1:0; flow rate 4 ml/min; detection at 215 nm;Chromolith SpeedROD C₁₈ column, 50×4.6 mm.

Gradient 6: linear gradient from over 1.75 min of MeCN/0.09% TFA andH₂O/0.1% TFA from 1:49 to 1:0 and 0.75 min at 1:0; flow rate 4 ml/min;detection at 215 nm; Chromolith SpeedROD C₁₈ column, 50×4.6 mm.

Gradient 7: linear gradient from over 2.5 min of MeCN/0.09% TFA andH₂O/0.1% TFA from 1:49 to 1:0; flow rate 4 ml/min; detection at 215 nm;UP50 DB-5M Uptisphere C₁₈ column, 5.0 μm, 50×4.6 mm.

Gradient 8: linear gradient over 7 min of MeCN/0.09% TFA and H₂O/0.1%TFA from 1:49 to 1:0 and 3 min at 1:0; flow rate 2.0 mL/min, detectionat 215 nm; SMT C₁₈ column, 5 μm, 250×4.6 mm.

Gradient 9: linear gradient over 10 min of MeCN/0.09% TFA and H₂O/0.1%TFA from 1:49 to 1:0; flow rate 2.0 mL/min, detection at 215 nm; SMT C₁₈column, 5 μm, 250×4.6 mm.

t_(R): retention time

NVP-AFC771-AI-17/CGE-3133

EXAMPLE 1Ac-Ala-Ala-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-His-NH₂TFA Salt

The title peptide is synthesised on a Milligen 9050 automated peptidesynthesizer (continuous flow; Millipore, Bedford, Mass., USA), startingwith an Fmoc-PAL-PEG-PS resin (see Albericio, F. et al, J. Org. Chem.,55 (1990) 3730-3743) for establishing the C-terminal carboxamide, andusing chemical protocols based on the fluorenylmethoxycarbonyl chemistry(see E. Atherton and R. C. Sheppard, in Solid-Phase Peptide Synthesis-APractical Approach, eds: R. Rickwood and B. D. Hames, IRL Press atOxford University Press, Oxford, 19989). The required Fmoc-amino acids(3 equiv.) are incorporated using their 2,4,5-trichlorophenyl esters(single coupling) with minimum reaction times of 30 min (see 9050 PlusPepSynthesizer User's Guide, Millipore Corporation, Bedford, Mass.,1992). Side chains are protected with the following groups: tert-butylfor aspartic acid, glutamic acid and serine;2,2,5,7,8-pentamethyl-chroman-6-sulfonyl for arginine; and trityl forhistidine. If required, a double coupling is performed using theFmoc-amino acid (3 equiv.) andO-(1,2-dihydro-2-oxo-1-pyridyl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (3 equiv.) in the presence of diisopropylethylamine (6equiv.). The complete peptide resin obtained after the final couplingreaction is simultaneously deprotected and cleaved by treatment withtrifluoroacetic acid/water (95:5, v/v) for 3 h at room temperature. Thefiltrate from the cleavage reaction is precipitated in diisopropylether-petroleum ether (1:1, v/v) at 0° C., and the precipitate iscollected by filtration. The crude peptide is purified by reversed-phasemedium-pressure liquid chromatography using a C₁₈ column eluted with anacetonitrile-water gradient containing 0.1% trifluoroacetic acid (MerckLICHROPREP RP-18, 15-25 μm bead diameter, reversed phase column materialbased on C₁₈-derivatised silicagel, Merck, Darmstadt, FRG; column length46 cm, diameter 3.6 cm; flow rate 53.3 ml/min; detection at 215 nm).Mass spectrometric analysis (matrix-assisted laser-desorption ionizationtime-of-flight mass spectrometry, MA/DI-TOF) of the purified compoundreveals molecular masses within 0.1% of the expected value (negative ionmode): 2463.3 (calc. 2463.7, C₁₀₄H₁₆₅N₃₆O₃₄). The purity of the titlecompound is verified by reversed-phase analytical HPLC: single peak att_(R)=1.81 min (Gradient 1); t_(R)=1.41 min (Gradient 2); and t_(R)=2.08min (Gradient 3).

The following peptides are synthesised as described in Example 1.

NVP-AFC759-AI-1/CGE-3132

EXAMPLE 2Ac-Ala-Asp-Ala-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-His-NH₂TFA Salt

Title compound: Mass spectral analysis (negative-ion mode): 2491.8(calc. 2491.7, C₁₀₅H₁₆₅N₃₆O₃₅),

Analytical HPLC: t_(R)=1.83 min (Gradient 1); t_(R)=1.41 min (Gradient2); and t_(R)=2.08 min (Gradient 3).

NVP-AFC757-AI-1/CGE-3131

EXAMPLE 3Ac-Ala-Asp-Ser-Ala-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-His-NH₂TFA Salt

Title compound: Mass spectral analysis (negative-ion mode): 2521.8(calc. 2521.7, C₁₀₆H₁₆₇N₃₆O₃₆),

Analytical HPLC: t_(R)=1.84 min (Gradient 1); t_(R)=1.42 min (Gradient2); and t_(R)=2.10 min (Gradient 3).

NVP-AFC756-AI-1/CGE-3130

EXAMPLE 4Ac-Ala-Asp-Ser-Gly-Ala-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-His-NH₂TFA

Title compound: Mass spectral analysis (negative-ion mode): 2449.6(calc. 2449.7, C₁₀₃H₁₆₃N₃₆O₃₄),

Analytical HPLC: t_(R)=1.84 min (Gradient 1); t_(R)=1.41 min (Gradient2); and t_(R)=2.09 min (Gradient 3).

NVP-AFC753-AI-1/CGE-3129

EXAMPLE 5Ac-Ala-Asp-Ser-Gly-Glu-Ala-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-His-NH₂TFA Salt

Title compound: Mass spectral analysis (negative-ion mode): 2522.0(calc. 2521.7, C₁₀₆H₁₆₇N₃₆O₃₆),

Analytical HPLC: t_(R)=1.85 min (Gradient 1); t_(R)=1.43 min (Gradient2); and t_(R)=2.11 min (Gradient 3).

NVP-AFC723-AI-1/CGE-3128

EXAMPLE 6Ac-Ala-Asp-Ser-Gly-Glu-Gly-Ala-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-Iis-NH₂TFA Salt

Title compound: Mass spectral analysis (negative-ion mode): 2463.1(calc. 2463.7, C₁₀₄H₁₆₅N₃₆O₃₄).

Analytical HPLC: t_(R)=1.82 min (Gradient 1); t_(R)=1.41 min (Gradient2); and t_(R)=2.07 min (Gradient 3).

NVP-AFC779-AI-1/CGE-3127

EXAMPLE 7Ac-Ala-AspSer-Gly-Glu-Gly-Asp-Ala-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-His-NH₂TFA Salt

Title compound: Mass spectral analysis (negative-ion mode): 2431.7(calc. 2431.6, C₉₉H₁₆₁N₃₆O₃₆),

Analytical HPLC: t_(R)=1.66 min (Gradient 1); and t_(R)=1.94 min(Gradient 3).

NVP-AFB832-AI-1/CGE-3121

EXAMPLE 8Ac-Ala-Asp-Ser-Gly-Glu-Gly-Asp-Phe-Ala-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-His-NH₂TFA Salt

Title compound: Mass spectral analysis (negative-ion mode): 2465.6(calc. 2465.6, C₁₀₂H₁₅₉N₃₆O₃₆),

Analytical HPLC: t_(R)=1.66 min (Gradient 1); and t_(R)=1.94 min(Gradient 3).

NVP-AFB828-AI-1/CGE-3120

EXAMPLE 9Ac-Ala-Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Ala-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-His-NH₂TFA Salt

Title compound: Mass spectral analysis (negative-ion mode): 2449.1(calc. 2449.7, C₁₀₃H₁₆₃N₃₆O₃₄).

Analytical HPLC: t_(R)=1.91 min (Gradient 1); t_(R)=1.83 min (Gradient4); and t_(R)=1.44 min (Gradient 5).

NVP-AFB826-AI-1/CGE-3119

EXAMPLE 10Ac-Ala-Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Ala-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-His-NH₂TFA Salt

Title compound: Mass spectral analysis (negative-ion mode): 2521.7(calc. 2521.7, C₁₀₆H₁₆₇N₃₆O₃₆).

Analytical HPLC: t_(R)=1.85 min (Gradient 1); t_(R)=1.88 min (Gradient4); and t_(R)=1.47 min (Gradient 5).

NVP-AFB823-AI-1/CGE-3115

EXAMPLE 11Ac-Ala-Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Ala-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-His-NH2TFA Salt

Title compound: Mass spectral analysis (negative-ion mode): 2521.7(calc. 2521.7, C₁₀₆H₁₆₇N₃₆O₃₆).

Analytical HPLC: t_(R)=1.82 min (Gradient 1); t_(R)=1.84 min (Gradient4); and t_(R)=1.44 min (Gradient 5).

NVP-AFB821-AI-1/CGE-3114

EXAMPLE 12Ac-Ala-Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Ala-Val-Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-His-NH₂TFA Salt

Title compound: Mass spectral analysis (negative-ion mode): 2521.7(calc. 2521.7, C₁₀₆H₁₆₇N₃₆O₃₆).

Analytical HPLC: t_(R)=1.80 min (Gradient 1); t_(R)=1.83 min (Gradient4); and t_(R)=1.43 min (Gradient 5).

NVP-AFB818-AI-1/CGE-3113

EXAMPLE 13Ac-Ala-Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Ala-Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-His-NH₂TFA Salt

Title compound: Mass spectral analysis (negative-ion mode): 2479.7(calc. 2479.7, C₁₀₃H₁₆₁N₃₆O₃₆).

Analytical HPLC: t_(R)=1.76 min (Gradient 1); t_(R)=1.78 min (Gradient4); and t_(R)=1.41 min (Gradient 5).

NVP-AFB504-AI-1/CGE-3110

EXAMPLE 14Ac-Ala-Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Ala-Gly-Pro-Arg-Val-Val-Glu-Arg-His-NH₂TFA Salt

Title compound: Mass spectral analysis (negative-ion mode): 2422.6(calc. 2422.6, C₁₀₂H₁₅₈N₃₃O₃₆).

Analytical HPLC: t_(R)=1.79 min (Gradient 4); and t_(R)=1.43 min(Gradient 5).

NVP-AFB503-AI-1/CGE-3109

EXAMPLE 15Ac-Ala-Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Ala-Pro-Arg-Val-Val-Glu-Arg-His-NH₂TFA Salt

Title compound: Mass spectral analysis (negative-ion mode): 2521.1(calc. 2521.7, C₁₀₆H₁₆₇N₃₆O₃₆).

Analytical HPLC: t_(R)=1.77 min (Gradient 4); and t_(R)=1.41 min(Gradient 5).

NVP-AFB502-AI-1/CGE-3108

EXAMPLE 16Ac-Ala-Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Ala-Arg-Val-Val-Glu-Arg-His-NH₂TFA Salt

Title compound: Mass spectral analysis (negative-ion mode): 2480.1(calc. 2481.7, C₁₀₃H₁₆₃N₃₆O₃₆).

Analytical HPLC: t_(R)=1.74 min (Gradient 4); and t_(R)=1.40 min(Gradient 5).

NVP-AFB501-AI-1/CGE-3105

EXAMPLE 17Ac-Ala-Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Ala-Val-Val-Glu-Arg-His-NH₂TFA Salt

Title compound: Mass spectral analysis (negative-ion mode): 2422.3(calc. 2422.6, C₁₀₂H₁₅₈N₃₃O₃₆).

Analytical HPLC: t_(R)=1.76 min (Gradient 1); t_(R)=1.79 min (Gradient4); and t_(R)=1.43 min (Gradient 5).

NVP-AFB500-AI-I/CGE-3104

EXAMPLE 18Ac-Ala-Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Ala-Val-Glu-ArgHis-NH₂TFA Salt

Title compound: Mass spectral analysis (negative-ion mode): 2481.0(calc. 2479.7, C₁₀₃H₁₆₁N₃₆O₃₆).

Analytical HPLC: t_(R)=1.72 min (Gradient 4); and t_(R)=1.38 min(Gradient 5).

NVP-AFB490-AI-1/CGE-3103

EXAMPLE 19Ac-Ala-Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Ala-Glu-Arg-His-NH₂TFA Salt

Title compound: Mass spectral analysis (negative-ion mode): 2481.7(calc. 2479.7, C₁₀₃H₁₆₁N₃₆O₃₆).

Analytical HPLC: t_(R)=1.72 min (Gradient 4); and tR-1.38 min (Gradient5).

NVP-AFB400-AI-1/CGE-3100

EXAMPLE 20Ac-Ala-Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Ala-Arg-His-NH₂TFA Salt

Title compound: Mass spectral analysis (negative-ion mode): 2450.0(calc. 2449.7, C₁₀₃H₁₆₃N₃₆O₃₄).

Analytical HPLC: t_(R)=1.77 min (Gradient 4); and t_(R)=1.40 min(Gradient 5).

NVP-AFB376-AI-1/CGE-3099

EXAMPLE 21Ac-Ala-Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Glu-Ala-His-NH₂TFA Salt

Title compound: Mass spectral analysis (negative-ion mode): 2421.0(calc. 2422.6, C₁₀₂H₁₅₈N₃₃O₃₆).

Analytical HPLC: t_(R)=1.78 min (Gradient 4); and t_(R)=1.43 min(Gradient 5).

NVP-AFB358-AI-1/CGE-3098

EXAMPLE 22Ac-Ala-Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-Ala-NH₂TFA Salt

Title compound: Mass spectral analysis (negative-ion mode): 2442.1(calc. 2441.7, C₁₀₂H₁₆₃N₃₄O₃₆).

Analytical HPLC: t_(R)=1.79 min (Gradient 4); and t_(R)=1.44 min(Gradient 5).

NVP-AEZ772-AI-1/2/CGE-2943

EXAMPLE 23Biotin-8-amino-3,6-dioxaoctanoyl-Ala-Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-His-NH₂TFA Salt

Fmoc-8-amino-3,6-dioxaoctanoic acid (3 equiv.; Neosystem, Strasbourg,France) is incorporated usingO-(1,2-dihydro-2-oxo-1-pyridyl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (3 equiv.; single coupling) in the presence ofdiisopropylethylamine (6 equiv.). (+)-Biotin (3 equiv.; Fluka, Buchs,Switzerland) is coupled usingO-(1,2-dihydro-2-oxo-1-pyridyl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (3 equiv.; first coupling) andN-[(dimethylamino)-1H-1,2,3,-triazolo-[4,5b]pyridin-1-yl-methylene]-N-methylmethanaminiumhexafluorophosphate N-oxide (3 equiv.; second coupling) in the presenceof diisopropylethyl amine (6 equiv.) Title compound: Mass spectralanalysis (negative-ion mode): 2837.1 (calc. 2837.1, C₁₁₉H₁₈₈N₃₉O₄₀S₁).

Analytical HPLC: t_(R)=1.74 min (Gradient 4); and t_(R)=1.42 min(Gradient 5).

NVP-AEZ775-AI-1/2/3/CGE-2944

EXAMPLE 24Fluorescein-8-amino-3,6-dioxaoctanoyl-Ala-Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-His-NH₂TFA Salt

Fmoc-8-amino-3,6-dioxaoctanoic acid (3 equiv.; Neosystem, Strasbourg,France) is incorporated usingO-(1,2-dihydro-2-oxo-1-pyridyl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (3 equiv.; single coupling) in the presence ofdiisopropylethylamine (6 equiv.). Fluoresceinisothiocyanate isomer I (3equiv.; Fluka, Buchs, Switzerland) is incorporated to the N-terminalamino group in the presence of diisopropylethylamine (6 equiv.).Coupling is achieved by dissolving the building block and the base inN-methyl-2-pyrrolidone, adding the mixture to the resin, and shaking atroom temperature for 21 h.

Title compound: Mass spectral analysis (negative-ion mode): 3000.6(calc. 3000.2, C₁₃₀H₁₈₅N₃₈O₄₃S₁).

Analytical HPLC: t_(R)=2.00 min (Gradient 4); and t_(R)=1.63 min(Gradient 5).

NVP-AEZ776-AI-1/CGE-2957

EXAMPLE 25 Ac-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-His-NH₂ TFASalt

Title compound: Mass spectral analysis (negative-ion mode): 1358.3(calc. 1358.6, C₅₇H₉₇N₂₄O₁₅).

Analytical HPLC: t_(R)=1.46 min (Gradient 4); and tR-1.13 min (Gradient6).

NVP-AEZ991-AI-11CGE-2959

EXAMPLE 26 Ac-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-His-NH₂TFA Salt

Title compound: Mass spectral analysis (negative-ion mode): 1415.8(calc. 1415.6, C₅₉H₁₀₀N₂₅O₁₆).

Analytical HPLC: t_(R)=1.45 min (Gradient 4); and t_(R)=1.11 min(Gradient 6).

NVP-AFA011-AI-1/CGE-2960

EXAMPLE 27Ac-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-His-NH₂ TFA Salt

Title compound: Mass spectral analysis (positive-ion mode): 1546.8(calc. 1546.7, C₆₋₄Hlo₉N₂₆O₁₉).

Analytical HPLC: t_(R)=1.45 min (Gradient 4); and t_(R)=1.11 min(Gradient 6).

NVP-AFA014-AI-1/CGE-2961

EXAMPLE 28Ac-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-His-NH2 TPASalt

Title compound: Mass spectral analysis (positive-ion mode): 1617.5(calc. 1617.8, C₆₇H₁₁₄N₂₇O₂₀).

Analytical HPLC: t_(R)=1.47 min (Gradient 4); and t_(R)=1.11 min(Gradient 6).

NVP-AFA017-AI-1/CGE-2962

EXAMPLE 29Ac-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-His-NH2TFA Salt

Title compound: Mass spectral analysis (positive-ion mode): 1731.6(calc. 1731.0, C₇₂H₁₂₅N₂₈O₂₁).

Analytical HPLC: t_(R)=1.65 min (Gradient 4); and t_(R)=1.21 min(Gradient 6).

NVP-AFA020-AI-1/CGE-2963

EXAMPLE 30Ac-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-His-NH2

Title compound: Mass spectral analysis (positive-ion mode): 1878.2(calc. 1878.2, C₈₂H₁₃₄N₂₉O₂₂).

Analytical HPLC: t_(R)=1.82 min (Gradient 4); and t_(R)=1.32 min(Gradient 6).

NVP-AFA022-AI-1/CGE-2964

EXAMPLE 31Ac-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-His-NH₂TFA Salt

Title compound: Mass spectral analysis (negative-ion mode): 1990.9(calc. 1991.2, C₈₆H₁₃₇N₃O₂₅).

Analytical HPLC: t_(R)=1.86 min (Gradient 4); and t_(R)=1.31 min(Gradient 6).

NVP-AFA023-AI-1/CGE-2965

EXAMPLE 32Ac-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-His-NH₂TFA Salt

Title compound: Mass spectral analysis (positive-ion mode): 2049.7(calc. 2050.3, C₈₈H₁₄₂N₃₁O₂₆).

Analytical HPLC: t_(R)=1.83 min (Gradient 4); and t_(R)=1.30 min(Gradient 6).

NVP-AFA027-AI-1/CGE-2966

EXAMPLE 33Ac-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-His-NH₂TFA Salt

Title compound: Mass spectral analysis (positive-ion mode): 2179.3(calc. 2179.4, C₉₃H₁₄₉N₃₂O₂₉).

Analytical HPLC: t_(R)=1.81 min (Gradient 4); and t_(R)=1.29 min(Gradient 6).

NVP-AFA030-AI-1/CGE-2967

EXAMPLE 34Ac-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-His-NH₂TFA Salt

Title compound: Mass spectral analysis (positive-ion mode): 2234.5(calc. 2234.5, C₉₅H₁₅₀N₃₃O₃₀).

Analytical HPLC: t_(R)=1.79 min (Gradient 4); and t_(R)=1.27 min(Gradient 6).

NVP-AFA031-AI-I/CGE-2968

EXAMPLE 35Ac-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-His-NH₂TFA Salt

Tide compound: Mass spectral analysis (negative-ion mode): 2321.0 (calc.2321.5, C₉₈H₁₅₅N₃₄O₃₂). Analytical HPLC: t_(R)=1.79 min (Gradient 4);and t_(R)=1.26 min (Gradient 6).

NVP-AFA036-AI-1/CGE-2969

EXAMPLE 36Ac-Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-His-NH₂TFA Salt

Title compound: Mass spectral analysis (negative-ion mode): 2436.3(calc. 2436.6, C₁₀₂H₁₆₀N₃₅O₃₅).

Analytical HPLC: t_(R)=1.78 min (Gradient 4); and t_(R)=1.27 min(Gradient 6).

NVP-AEZ570-AI-1/CGE-2809

EXAMPLE 37Ac-Ala-Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-NH2TFA Salt

Title compound: Mass spectral analysis (negative-ion mode): 1576.5(calc. 1576.6, C₆₅H₉₉N₂₀O₂₆).

Analytical HPLC: t_(R)=1.52 min (Gradient 7); and t_(R)=5.08 min(Gradient 8).

NVP-AEZ572-AI-1/2/CGE-2810

EXAMPLE 38Ac-Ala-Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-His-NH₂TFA Salt

Title compound: Mass spectral analysis (negative-ion mode): 2507.7(calc. 2507.7, C₁₀₅H₁₆₅N₃₆O₃₆).

Analytical HPLC: t_(R)=1.87 min (Gradient 4); and t_(R)=1.49 min(Gradient 5).

NVP-AEZ575-AI-1/CGE-2811

EXAMPLE 39Ac-Ala-Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-His-Gln-Ser-Ala-Cys-NH₂TFA Salt

Title compound: Mass spectral analysis (negative-ion mode): 2898.0(calc. 2897.1, C₁₁₉H₁₈₈N₄₁O₄₂S₁).

Analytical HPLC: t_(R)=5.01 min (Gradient 8); and t_(R)=5.76 min(Gradient 9).

NVP-AEZ576-AI-1/CGE-2827

EXAMPLE 40(Ac-Ala-Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-His-Gln-Ser-Ala-Cys-NH₂)SS(Ac-Ala-Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-His-Gln-Ser-Ala-Cys-NH₂)TEA Salt

The peptide (50 mg), which is described in Example 39, is dissolved in a1% solution of sodium carbonate (500 μl) and stirred at room temperatureovernight. After this time, the crude peptide purified as indicated inExample 1.

Title compound: Mass spectral analysis (negative-ion mode): 5795.1(calc. 5793.3, C₂₃₈H₃₇₅N₈₂O₈₄S₂).

Analytical HPLC: t_(R)=1.98 min (Gradient 4); and t_(R)=1.56 min(Gradient 5).

NVP-AHI361-AI-1/CGE-3348

EXAMPLE 41Ac-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-His-NH2 TFASalt

Title compound: Mass spectral analysis (negative-ion mode): 1615.8(calc. 1615.8, C₆₇H₁₁₂N₂₇O₂₀).

Analytical HPLC: t_(R)=1.58 min (Gradient 4); and t_(R)=1.29 min(Gradient 5).

NVP-AHI366-AI-1/CGE-3349

EXAMPLE 42Ac-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-NH₂ TFASalt

Title compound: Mass spectral analysis (negative-ion mode): 1591.4(calc. 1591.8, C₆₇H₁ 16N₂₅O₂₀).

Analytical HPLC: t_(R)=1.76 min (Gradient 4); and t_(R)=1.37 min(Gradient 5).

NVP-AHI378-AI-I/CGE-3350

EXAMPLE 43Ac-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Glu-NH2 TFASalt

Title compound: Mass spectral analysis (negative-ion mode): 1582.6(calc. 1582.8, C₇₀H₁₁₃N₂₂O₂₀).

Analytical HPLC: t_(R)=2.03 min (Gradient 4); and t_(R)=1.59 min(Gradient 5).

NVP-AHI396-AI-1/CGE-3351

EXAMPLE 44Ac-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-NH₂ TFASalt

Title compound: Mass spectral analysis (negative-ion mode): 1568.0(calc. 1568.8, C₆₉H₁₁₁N₂₂O₂₀).

Analytical HPLC: t_(R)=2.03 min (Gradient 4); and t_(R)=1.55 min(Gradient 5).

NVP-AHI398-AI-1/CGE-3352

EXAMPLE 45Ac-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-NH2 TFASalt

Title compound: Mass spectral analysis (negative-ion mode): 1526.5(calc. 1526.7, C₆₆H₁₁O₅N₂₂O₂₀).

Analytical HPLC: t_(R)=1.96 min (Gradient 4); and t_(R)=1.50 min(Gradient 5).

NVP-AHI399-AI-1/CGE-3353

EXAMPLE 46Ac-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-NH2 TFASalt

Title compound: Mass spectral analysis (negative-ion mode): 1556.3(calc. 1556.7, C₆₇H₁₀₅N₂₁O₂₂).

Analytical HPLC: t_(R)=1.86 min (Gradient 4); and t_(R)=1.48 min(Gradient 5).

NVP-AHI400-AI-1/CGE-3354

EXAMPLE 47Ac-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-NH2 TFASalt

Title compound: Mass spectral analysis (negative-ion mode): 1457.3(calc. 1457.6, C₆₂H₉₄N₁₉O₂₂).

Analytical HPLC: t_(R)=1.89 min (Gradient 4); and t_(R)=1.50 min(Gradient 5).

NVP-AHI401-AI-1/CGE-3355

EXAMPLE 48Ac-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-NH₂ TFASalt

Title compound: Mass spectral analysis (negative-ion mode): 1447.3(calc. 1447.5, C₆₀H₉₂N₁₉O₂₃).

Analytical HPLC: t_(R)=1.83 min (Gradient 4); and t_(R)=1.46 min(Gradient 5).

NVP-AHI402-AI-1/CGE-3356

EXAMPLE 49Ac-Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-NH2 TFASalt

Title compound: Mass spectral analysis (negative-ion mode): 1505.1(calc. 1505.6, C₆₂H₉₄N₁₉O₂₅).

Analytical HPLC: t_(R)=1.84 min (Gradient 4); and t_(R)=1.49 min(Gradient 5).

NVP-AHI793-AI-1/CGE-3357

EXAMPLE 50Ac-Ala-Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-NH2 TFASalt

Title compound: Mass spectral analysis (negative-ion mode): 1420.6(calc. 1420.5, C₅₉H₈₇N₁₆O₂₅).

Analytical HPLC: t_(R)=1.86 min (Gradient 4); and t_(R)=1.48 min(Gradient 5).

NVP-ANH419-AI-1, CGE-3459

EXAMPLE 51 Ac-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-NH₂ TFA Salt

Title compound: Mass spectral analysis (negative-ion mode): 1117.2(calc. 1117.2, C₄₈H₇₄N₁₅O₁₆).

Analytical HPLC, t_(R)=1.94 min (Gradient 4); t_(R)=1.58 min (Gradient5).

NVP-AML639-NX-1, CGE-3639

EXAMPLE 52 Ac-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-NH₂

Title compound: Mass spectral analysis (negative-ion mode): 903.6 (calc.904.0, C₄₀H₅₉N₁₀O₁₄).

Analytical HPLC, t_(R)=1.97 min (Gradient 4); t_(R)=1.56 min (Gradient5).

NVP-AML640-NX-1, CGE-3640

EXAMPLE 53 Ac-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-NH₂

Title compound: Mass spectral analysis (negative-ion mode): 804.5 (calc.804.8, C₃₅H₅₀N₉O₁₃).

Analytical HPLC, t_(R)=1.87 min (Gradient 4); t_(R)=1.50 min (Gradient5).

NVP-AML648-AI-1, CGE-3650

EXAMPLE 54 Ac-Asp-Phe-Leu-Ala-Glu-NHCH₂CH₂OCH₂CH₂OCH₂CO-Val-Arg-Gly-NH2TFA Salt

Fmoc-8-amino-3,6-dioxaoctanoic acid is purchased from Neosystem(Strasbourg, France). Title compound: Mass spectral analysis(negative-ion mode): 1090.7 (calc. 1091.2, C₄₈H₇₆N₁₃O₁₆).

Analytical HPLC, t_(R)=2.03 min (Gradient 4); t_(R)=1.59 min (Gradient5).

NVP-AML655-A-1, CGE-3651

EXAMPLE 55 Ac-Asp-Phe-Leu-Ala-Glu-Ala-Val-Arg-Gly-NH₂ TFA Salt

Fmoc-β-alanine is purchased from Fluka (Buchs, Switzerland). Titlecompound:

Mass spectral analysis (negative-ion mode): 1017.4 (calc. 1017.1,C₄₅H₇₀N₁₃O₁₄).

Analytical HPLC, t_(R)=1.99 min (Gradient 4); t_(R)=1.57 min (Gradient5).

NVP-AML658-AI-1, CGE-3652

EXAMPLE 56 Ac-Asp-Phe-Leu-Ala-Glu-Ala-Ala-Val-Arg-Gly-NH₂ TFA Salt

Fmoc-β-alanine is purchased from Fluka (Buchs, Switzerland). Titlecompound:

Mass spectral analysis (negative-ion mode): 1088.4 (calc. 1088.2,C₄₈H₇₅N₁₄O₁₅).

Analytical HPLC, t_(R)=1.95 min (Gradient 4); t_(R)=1.54 min (Gradient5).

NVP-AML632-AI-1 CGE-3638

EXAMPLE 57 Ac-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-NH₂ TFA Salt

Title compound: Mass spectral analysis (negative-ion mode): 1060.1(calc. 1060.2, C₄₆H₇₁N₁₄O₁₅).

Analytical HPLC, t_(R)=1.94 min (Gradient 4); t_(R)=1.54 min (Gradient5).

Tubule Formation Assay.

24 well plates were coated with 30 μl/well of growth factor-reduced(GF-reduced) Matrigel (Becton Dickinson Labware, Bedford, Mass.).Endothelial cells plated on this matrix migrate and differentiate intotubules within 6h of plating as described previously (14). HuDMECs orSVEC 4-10 cells were seeded at a density of 4×10⁴ cells/ml and incubatedfor 6h in 500 μl of either DMEM+1% FCS alone (control), or thismedium±10 ng/ml VEGF or bFGF in the presence or absence of fibrinogenE-fragment, fibrin E-fragment, FpA or α1-24. Assessment of tubuleformation involved fixing the cell preparation in 70% ethanol at 4° C.for 15 minutes, rinsing in PBS and staining with haematoxylin and eosin.Three random fields of view in 3 replicate wells for each test conditionwere visualised under low power (×40 magnification), and colour imagescaptured using a Fuji digital camera linked to a Pentium III computer(containing a frame grabber board). Tubule formation was assessed bycounting the number of tubule branches and the total area covered bytubules in each field of view using image analysis software supplied byScion Image.

Migration Assay

The Boyden chamber technique was adapted from (13) and used to evaluateHuDMEC migration across a porous membrane towards a concentrationgradient of either VEGF (10 ng/ml) or bFGF (10 ng/ml). The Neuro Probe48 well microchemotaxis chamber (Neuro Probe Inc, Cabin John, Md.) wasused with 8 μm pore size polycarbonate membranes (Neuro Probe Inc, CabinJohn, Md.) coated with 100 μg/ml collagen type IV. 10 ng/ml VEGF or bFGFalone or with various concentrations of fibrinogen E-fragment, fibrinE-fragment, FpA or α1-24 were dissolved in DMEM+1% FCS and placed in thelower wells. The collagen-coated membrane was then placed over this and5 μl of 25×10⁴ HuDMECs/ml (in DMEM containing 1% FCS) added to the upperchamber. The chambers were then incubated at 37° C. for 4.5h. Thechamber was then dismantled, the membrane removed and non-migrated cellsscraped off the upper surface. Migrated cells on the lower surface werefixed with methanol, stained with Hema ‘Gurr’ rapid staining kit (Merck,Leics, United Kingdom) and counted using a light microscope (×160magnification) in 3 random fields per well. Each test condition wascarried out in 3-6 replicate wells and each experiment repeated 3 times.

Proliferation Assay

The MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide)assay was used as previously described (12) to assess HuDMECproli0feration induced by VEGF or bFGF in the absence or presence offibrinogen E-fragment, fibrin E-fragment, FpA or α1-24. HuDMEC wereseeded at 3×10³ cells/100 in DMEM+1% FCS±10 ng/ml VEGF or bFGF in testsolution into 96 well microtitre plate for 4.5 and 6h. At these timepoints, a quarter volume of MTT solution (2 mg MTT/ml PBS) was added toeach well and each plate was incubated for 4h at 37° C. resulting in aninsoluble purple formazan product. The medium was aspirated and theprecipitates dissolved in 1001 Σl DMSO buffered at pH 10.5. Theabsorbance was then read at 540 nm on a Dynex ELISA plate reader.

Cytotoxicity Assay

HuDMECs were seeded at a density of 1-2×10⁵ cells per well in a 24well-plate in the absence or presence of fibrinogen E-fragment, fibrinE-fragment, FpA or α1-24. After 6h, both live (following removal bytrypsinisation) and dead (floating) cells were harvested and cellviability of all cells present assessed using propidium iodide stainingof 5000 cells in each of triplicate samples per treatment using aFACScan (Becton Dickinson) equipped with a blue laser excitation of 15mW at 488 nm. The data was collected and analysed using Cell Questsoftware (Becton Dickinson).

In vivo Efficacy of Peptides

Experiments were performed on six-week-old Balb/C mice weighing 15 g,obtained from Sheffield Field Laboratories. All experiments wereapproved by the Home Office Project Licence Number PPL50/1414.

Tumour Cell Culture

The CT26 cell line was maintained by in vitro passage in Dulbecco'sMinimal Eagles Medium containing 10% foetal calf serum, and 1%penicillin and streptomycin and maintained at 37° C. in humidifiedatmosphere of 5% CO₂ in air. The cell line was routinely checked toensure freedom from mycoplasma (Mycoplasma rapid detection system,Gena-Probe Incorporated, U.S.A.).

Subcutaneous Tumour Implantation

Animals were anaesthetised with an intraperitoneal injection of diazepam(0.5 mg/ml, Dumex Ltd.) and hypnorm (fentanyl citrate 0.0315 mg/ml andfluanisone 1 mg/ml, Janssen Pharmaceutical Ltd.) in the ratio of 1:1 ata volume of 0.1 ml/200 g body weight, with supplementation as requiredto maintain adequate anaesthesia. Naïve Balb/c mice were immunised s.cinto the right flank, following removal of the fur.

Tumour cells were injected at a concentration of 3×10⁵ viable CT26 cellsper animal suspended in 100 ul serum free medium. Animals were thenallowed to recover.

Tumour growth and animal weights were monitored daily.

Administration of Peptides

Tumour growth was measured daily and when the majority of animals in thecohort had tumour volumes of>100 mm³ but<350 mm³ animals were dividedinto experimental and control groups. This occurred between 14 and 18days following implantation of the tumour cell suspension. Animals thenreceived an intraperitoneal (ip) injection of either active drug(peptide α1-24 100 mM; 1001 μl) or vehicle (phophate buffered saline,100 μl). Daily injections continued until the tumour growth in thecontrol animals reached the maximum burden allowed by Home Officelegislation.

Assessment of Tumour Growth

Tumour volumes were assessed by calliper measurements of theperpendicular diameters and volumes estimated using the equation:—Volume=(a ² ×b)/2where a is the smaller and b the larger diameter

Animals were weighed on a daily basis and the general well beingmonitored.

Statistical Analysis.

All experiments were performed at least three times and data analysedusing the Mann-Whitney U test, a non-parametric test that does notassume a Gaussian distribution in the data being analysed. P<0.05 wastaken as significant.

References

-   1. Folkman J Angiogenesis in cancer, vascular, rheumatoid and other    disease. Nature Medicine, 1: 27-31, 1995.-   2. Leek R, Harris A L, and Lewis CE Cytokine networks in solid human    tumours: regulation of angiogenesis. J. Leuk. Biol., 56: 423-35,    1994.-   3. Cao Y Endogenous angiogenesis inhibitors: angiostatin,    endostatin, and other proteolytic fragments. Prog Mol Subcell Biol.,    20:161-76, 1998.-   4. Doolittle R Fibrinogen and Fibrin. Scientific American, 245:    92-101, 1981.-   5. Costantini V, Zacharski L R, Memoli V A, Kisiel W, Kudryk B J,    and Rousseau S M Fibrinogen deposition without thrombin generation    in primary human breast cancer tissue. Cancer Res., 51:349-53, 1991.-   6. Dvorak H F, Nagy J A, Feng D, Brown L F, and Dvorak A M Vascular    permeability factor/vascular endothelial growth factor and the    significance of microvascular hyperpermeability in angiogenesis.    Curr Top Microbiol Immunol, 237:97-132, 1999.-   7. Thompson W D, Wnag J E H, Wilson S J, and Ganesalinghiam N    Angiogenesis and fibrin degradation in human breast cancer.    Angiogenesis: Molecular Biology, Clinical Aspects, 245-251, 1994.-   8. Thompson W D, Smith E B, Stirk C M, Marshall F I, Stout A J, and    Kocchar A Angiogenic activity of fibrin degradation products is    located in fibrin fragment E. J. Pathol, 168: 47-53, 1992.-   9. Malinda K M, Ponce L, Kleinman H K, Shackelton L M, and Millis A    J Gp38k, a protein synthesized by vascular smooth muscle cells,    stimulates directional migration of human umbilical vein endothelial    cells. Exp Cell Res 250:168-73, 1999.-   10. Shen J, Ham R G, Karmiol S Expression of adhesion molecules in    cultured human pulmonary microvascular endothelial cells. Microvasc    Res., 50:360-72, 1995.-   11. Liu J. Kolath J. Anderson J. Kolar C, Lawson TA, Talmadge J. and    Gmeiner W H Positive interaction between 5-FU and FdUMP[10] in the    inhibition of human colorectal tumour cell proliferation. Antisense    Nucleic Acid Drug Dev., 9(5):481-6, 1999.-   12. Dejano E, Languino L R, Polentarutti N, Balconi G, Ryckewaert J    J, Larrieu M J, Donati M B, Mantovani A, and Marguerie G Interaction    between fibrinogen and cultured endothelial cells. J. Clin. Invest.,    75: 11-18, 1985.-   13. Bootle-Wilbraham C A, Tazzyman S, Marshall J M, Lewis C E.    Fibrinogen E-fragment inhibits the migration and tubule formation of    human dermal microvascular endothelial cells in vitro. Cancer    Research (2000) 60: 4719-4724-   14. Marsh H C, Meinwald Y C, Lee S, Martinelli R A, Scheraga H A.    Mechanism of action of thrombin on fibrinogen: NMR evidence for a    beta-bend at or near fibrinogen A alpha Gly(P5)-Gly(P4).    Biochemistry (1985) 24: 2806-2812.-   15. Gellman S. H., Acc. Chem. Res. (1998)31: 173-180.

1. A polypeptide of 15 or less amino acid residues comprising a sequenceselected from the group consisting of: X₁FLAEX₆X₇X₈V DX₂LAEX₆X₇X₈VDFX₃AEX₆X₇X₈V DFLX₄EX₆X₇X₈V DFLAX₅X₆X₇X₈V DFLAEX₆X₇X₈V DFLAEX₆X₇X₈X₉

wherein X is any proteinogenic or non-proteinogenic amino acid residueand which polypeptide has anti-angiogenic activity.
 2. A polypeptideaccording to claim 1 wherein one or more amino acids are replaced by aconservative proteinogenic or non-proteinogenic amino acid.
 3. Apolypeptide according to claim 1 wherein X₁ is an acidic amino acid orwherein X₂, X₃, X₄, X₅ are selected from the groups consisting ofglycine or alanine, valine, leucine, isoleucine, proline, wherein X₆,X₇, X₈ any amino acid and wherein X₉ is a non-polar amino acid.
 4. Apolypeptide according to claim 1 wherein X₁ is asparatic acid, X₂ isphenylalanine, X₃ is leucine, X₄ is alanine, X₅ is glutamic acid, X₆,X₇, and X₈ are glycine, and X₉ is valine.
 5. A polypeptide according tothe previous claims wherein one or more of amino acid residues X₁ and X₅are replaced by an acidic non-proteinogenic amino acid, preferablyselected from the group consisting of: gamma-carboxy-L-glutamic acid,L-2-aminoadipic acid, L-3-aminoadipic acid and(+/−)-1minocyclopentane-cis-1,3-dicarboxylic acid.
 6. A polypeptideaccording to the previous-claims wherein X₂ is selected from the groupconsisting of L-2-naphthylalanine,L-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid and4,4′-biphenylalanine.
 7. A polypeptide according to the previous claimswherein one or more of amino acid residues X₃, X₄ or X₉ are replaced bya hydrophobic non-proteinogenic amino acid, preferably selected from thegroup consisting of 1-aminocyclopropanecarboxylic acid,3-aminopentane-3-carboxylic acid, R-2-amino-2-cyclohexyl-propanoic acid.8. A polypeptide according to the previous claims comprising a sequenceselected from the group consisting of: GX₁X₂X₃X₄X₅X₆X₇X₈X₉EGX₁X₂X₃X₄X₅X₆X₇X₈X₉ GEGX₁X₂X₃X₄X₅X₆X₇X₈X₉

wherein X₁, X₂, X₃, X₄, X₅, X₆, X₇, X₈ and X₉, respectively, are definedas above.
 9. A polypeptide according to the previous claims comprising asequence selected from the group consisting of: X₁X₂X₃X₄X₅X₆X₇X₈X₉RX₁X₂X₃X₄X₅X₆X₇X₈X₉RG X₁X₂X₃X₄X₅X₆X₇X₈X₉RGP

wherein X₁, X₂, X₃, X₄, X₅, X₆, X₇, X₈ and X₉, respectively, are definedas above.
 10. A polypeptide according to the previous claim wherein oneor more of residues X₇X₈X₉ are replaced by a flexible chemical linker.11. A polypeptide according to claim 10 wherein said linker has abackbone of comprises 3 to 40 atoms.
 12. The polypeptide of thepreceding claim wherein said polypeptide has a length of 9, 10, 11, 12,13, or 14 amino acid residues.
 13. A polypeptide according to claim 9which polypeptide comprises an amino acid sequence of the sequenceX_(a)FLAEGGGVX_(b)G, wherein X is any amino acid.
 14. The polypeptide ofclaim 13 wherein X_(a) is an acidic amino acid and X_(b) is a basicamino acid.
 15. The polypeptide of claim 14 wherein X_(b) is selectedfrom the group consisting of L-2,4-diaminobutyric acid, R-aminocamitine,L-alpha-amino-gamma-guanidinobutyric acid and omithine.
 16. Apolypeptide selected from the group consisting of: i) a peptide of thesequence, XXXXXLXEXXGXXXPRVXXR,

or part thereof, wherein X is any amino acid residue; ii) a peptide asrepresented in (i) wherein amino acid residue X is selected from thefollowing group: glycine, alanine, valine, leucine, isoleucine, proline;and iii) a peptide represented in (i) or (ii) which has anti-angiogenicactivity.
 17. A polypeptide according to any of claim 16 whichpolypeptide consists of an amino acid sequence selected from the groupconsisting of: GEG DFL AEG GGV RGP RVVE R GEG DFL AEG GGX RGP RVVE R GEGDFL AEG GGV XGP RVVE R GEG DFL AEG GGV RXP RVVE R GEG DFL AEG GGV RGPRVXE R GEG DFL AEG GGV RGP RVVXR GEG DFL AEG GGXXXP RVVX R GEG DFL AEGGGXXXP RVXXR.

wherein X is any amino acid residue.
 18. A polypeptide according toclaim 16 wherein X is an amino acid residue selected from the groupconsisting of glycine, alanine, valine, leucine, isoleucine, or proline.19. A polypeptide according to any of the previous claims whichpolypeptide is modified at the N-terminus.
 20. A polypeptide accordingto claim 19 wherein said modification is selected from the groupconsisting of carboxamide, carbamate, urea, sulphonamide, acetylationand alkylation.
 21. A polypeptide according to the previous claims whichpolypeptide is modified at the C-terminus.
 22. A polypeptide accordingto claim 21 wherein said modification is selected from the groupconsisting of amidate, carboxylic acid, ester, carboxamide and alcohol.23. A polypeptide according to the previous claims-which polypeptide ismodified at the N-terminus and at the C-terminus.
 24. A polypeptideaccording to the previous claims which polypeptide has one or moreL-amino acid replaced by the corresponding D-amino acid.
 25. Apolypeptide according to the previous claims which polypeptide has oneor more amino-acids are replaced by an α-methyl derivative and/or anN-methyl derivative.
 26. A polypeptide according to the previous claimwherein said polypeptide is modified by cyclisation.
 27. A polypeptideaccording to claim 26 wherein said cyclisation is a head to tailcyclisation, side-chain to side-chain cyclisation, side-chain to endcyclisation, branched cyclisation or backbone to backbone cyclisation.28. A polypeptide according to the previous claims wherein saidpolypeptide comprises one or more chemical modifications of thebackbone.
 29. A polypeptide according to claim 28 wherein said modifiedbackbone is selected from the group consisting of: beta-peptidebackbone, depsipeptide backbone, oligosulfonamide backbone, oliogureaand thiourea backbone, oligocarbamate backbone, peptoid backbone andazapeptide backbone.
 30. The polypeptide of the preceding claims-whereinsaid polypeptide consists of said sequence.
 31. The polypeptide of thepreceding claims wherein said polypeptide inhibits cell proliferation byat least 10 percent relative to a control without said polypeptide in aproliferation assay.
 32. A polypeptide according to claim wherein saidpolypeptide inhibits tumour growth by at least 10 percent relative to acontrol without said polypeptide.
 33. A pharmaceutical compositioncomprising a polypeptide according to claim
 1. 34. A pharmaceuticalcomposition comprising two or more polypeptides according to claim 1wherein said agent has anti-angiogenic activity.
 35. A pharmaceuticalcomposition according to claim 34 wherein said two or more polypeptidesare linked by a linker molecule.
 36. A pharmaceutical compositionaccording to claim 34 wherein said pharmaceutical composition comprisesa plurality of polypeptides.
 37. A pharmaceutical composition accordingto claim 34 wherein said pharmaceutical composition comprises 3, 4, 5,6, 7, 8, 9, or 10 polypeptides linked together as an oligomericpolypeptide.
 38. A pharmaceutical composition according to claim 34wherein said pharmaceutical composition is a dimer of two polypeptides.39. Use of a polypeptide according to claim 1 for the manufacture of amedicament for use in the treatment of cancer.
 40. An isolated nucleicacid molecule consisting of a DNA sequence encoding a polypeptideaccording to claim
 1. 41. An isolated nucleic acid molecule whichanneals under stringent hybridisation conditions to the sequenceaccording to claim
 40. 42. A vector comprising a nucleic acid moleculeaccording to claim 40 operably linked to a promoter.
 43. A vectoraccording to claim 42 which is an expression vector adapted forprokaryotic or eukaryotic cell expression.
 44. A celltransformed/transfected with the nucleic acid according to claim 40 orthe vector according to claim
 42. 45. A method for the production ofpolypeptides according to claim 1: i) providing a cell according toclaim 44; ii) providing conditions conducive to the manufacture of saidpolypeptides; and iii) purifying said polypeptides from a cell, or acells culture environment.
 46. A non-human, transgenic animalcharacterised in that said animal incorporates a nucleic acid moleculeencoding a polypeptide according to of claim
 1. 47. A method to treat ananimal which would benefit from inhibition of angiogenesis comprising:i) administering an effective amount of a polypeptide according to claim1 to an animal to be treated; ii). monitoring the effects of saidpolypeptide on the inhibition of angiogenesis.
 48. A method to treat ananimal which would benefit from inhibition of angiogenesis comprising:i) administering an effective amount of an pharmaceutical compositionaccording to claim 33 to an animal to be treated; ii). monitoring theeffects of said agent on the inhibition of angiogenesis.
 49. A method totreat an animal which would benefit from inhibition of angiogenesiscomprising: i) administering an effective amount of a nucleic acidmolecule according to claim 40 or a vector according to claim 42 to ananimal to be treated; ii). monitoring the effects of said nucleic acidor vector on the inhibition of angiogenesis.
 50. An imaging agentcomprising a polypeptide according to any of claim 1.